A simple method to generate human airway epithelial organoids with externally orientated apical membranes

Organoids, which are self-organizing three-dimensional cultures, provide models that replicate specific cellular components of native tissues or facets of organ complexity. We describe a simple method to generate organoid cultures using isolated human tracheobronchial epithelial cells grown in mixed matrix components and supplemented at day 14 with the Wnt pathway agonist R-spondin 2 (RSPO2) and the bone morphogenic protein antagonist Noggin. In contrast to previous reports, our method produces differentiated tracheobronchospheres with externally orientated apical membranes without pretreatments, providing an epithelial model to study cilia formation and function, disease pathogenesis, and interaction of pathogens with the respiratory mucosa. Starting from 3 × 10⁵ cells, organoid yield at day 28 was 1,720 ± 302. Immunocytochemistry confirmed the cellular localization of airway epithelial markers, including CFTR, Na⁺/K⁺ ATPase, acetylated-α-tubulin, E-cadherin, and ZO-1. Compared to native tissues, expression of genes related to bronchial differentiation and ion transport were similar in organoid and air-liquid interface (ALI) cultures. In matched primary cultures, mean organoid cilia length was 6.1 ± 0.2 µm, similar to that of 5.7 ± 0.1 µm in ALI cultures, and ciliary beating was vigorous and coordinated with frequencies of 7.7 ± 0.3 Hz in organoid cultures and 5.3 ± 0.8 Hz in ALI cultures. Functional measurement of osmotically induced volume changes in organoids showed low water permeability. The generation of numerous single testable units from minimal starting material complements prior techniques. This culture system may be useful for studying airway biology and pathophysiology, aiding diagnosis of ciliopathies, and potentially for high-throughput drug screening.